Bone marrow failure unresponsive to bone marrow transplant is caused by mutations in thrombopoietin.

We report 5 individuals in 3 unrelated families with severe thrombocytopenia progressing to trilineage bone marrow failure (BMF). Four of the children received hematopoietic stem cell transplants and all showed poor graft function with persistent severe cytopenias even after repeated transplants with different donors. Exome and targeted sequencing identified mutations in the gene encoding thrombopoietin (THPO): THPO R99W, homozygous in affected children in 2 families, and THPO R157X, homozygous in the affected child in the third family. Both mutations result in a lack of THPO in the patients' serum. For the 2 surviving patients, improvement in trilineage hematopoiesis was achieved following treatment with a THPO receptor agonist. These studies demonstrate that biallelic loss-of-function mutations in THPO cause BMF, which is unresponsive to transplant due to a hematopoietic cell-extrinsic mechanism. These studies provide further support for the critical role of the MPL-THPO pathway in hematopoiesis and highlight the importance of accurate genetic diagnosis to inform treatment decisions for BMF.

Genetic features of myelodysplastic syndrome and aplastic anemia in pediatric and young adult patients.

The clinical and histopathological distinctions between inherited versus acquired bone marrow failure and myelodysplastic syndromes are challenging. The identification of inherited bone marrow failure/myelodysplastic syndromes is critical to inform appropriate clinical management. To investigate whether a subset of pediatric and young adults undergoing transplant for aplastic anemia or myelodysplastic syndrome have germline mutations in bone marrow failure/myelodysplastic syndrome genes, we performed a targeted genetic screen of samples obtained between 1990-2012 from children and young adults with aplastic anemia or myelodysplastic syndrome transplanted at the Fred Hutchinson Cancer Research Center. Mutations in inherited bone marrow failure/myelodysplastic syndrome genes were found in 5.1% (5/98) of aplastic anemia patients and 13.6% (15/110) of myelodysplastic syndrome patients. While the majority of mutations were constitutional, a RUNX1 mutation present in the peripheral blood at a 51% variant allele fraction was confirmed to be somatically acquired in one myelodysplastic syndrome patient. This highlights the importance of distinguishing germline versus somatic mutations by sequencing DNA from a second tissue or from parents. Pathological mutations were present in DKC1, MPL, and TP53 among the aplastic anemia cohort, and in FANCA, GATA2, MPL, RTEL1, RUNX1, SBDS, TERT, TINF2, and TP53 among the myelodysplastic syndrome cohort. Family history or physical examination failed to reliably predict the presence of germline mutations. This study shows that while any single specific bone marrow failure/myelodysplastic syndrome genetic disorder is rare, screening for these disorders in aggregate identifies a significant subset of patients with inherited bone marrow failure/myelodysplastic syndrome.

Targeted mass spectrometry enables robust quantification of FANCD2 mono-ubiquitination in response to DNA damage.

The Fanconi anemia pathway is an important coordinator of DNA repair pathways and is particularly relevant to repair of DNA inter-strand crosslinks. Central to the pathway is monoubiquitination of FANCD2, requiring the function of multiple proteins in an upstream Fanconi core complex. We present development and analytical characterization of a novel assay for quantification of unmodified and monoubiquitinated FANCD2 proteoforms, based on peptide immunoaffinity enrichment and targeted multiple reaction monitoring mass spectrometry (immuno-MRM). The immuno-MRM assay is analytically characterized using fit-for-purpose method validation. The assay linear range is >3 orders of magnitude with total repeatability <16% CV. In proof-of-principle experiments, we demonstrate application of the multiplex assay by quantifying the FANCD2 proteoforms following mitomycin-c treatment in an isogenic pair of FancA-corrected and uncorrected cell lines, as well as primary peripheral blood mononuclear cells from Fanconi Anemia patients. Additionally, we demonstrate detection of endogenous FANCD2 monoubiquitination in human breast cancer tissue. The immuno-MRM assay provides a potential functional diagnostic for patients with Fanconi Anemia with defects in the upstream FA complex or FANCD2, and a potential test for predicting sensitivity to DNA cross-linking agents in human cancers.

The eIF2-alpha kinase HRI is a novel therapeutic target in multiple myeloma.

Dexamethasone (dex) induces apoptosis in multiple myeloma (MM) cells and is a frontline treatment for this disease. However resistance to dex remains a major challenge and novel treatment approaches are needed. We hypothesized that dex utilizes translational pathways to promote apoptosis in MM and that specific targeting of these pathways could overcome dex-resistance. Global unbiased profiling of mRNA translational profiles in MM cells treated with or without dex revealed that dex significantly repressed eIF2 signaling, an important pathway for regulating ternary complex formation and protein synthesis. We demonstrate that dex induces the phosphorylation of eIF2α resulting in the translational upregulation of ATF4, a known eIF2 regulated mRNA. Pharmacologic induction of eIF2α phosphorylation via activation of the heme-regulated eIF2α kinase (HRI) induced apoptosis in MM cell lines and in primary MM cells from patients with dex-resistant disease. In addition, co-culture with marrow stroma failed to protect MM cells from apoptosis induced by targeting the eIF2 pathway. Combination therapy with rapamycin, an mTOR inhibitor, and BTdCPU, an activator of HRI, demonstrated additive effects on apoptosis in dex-resistant cells. Thus, specific activation of the eIF2α kinase HRI is a novel therapeutic target in MM that can augment current treatment strategies.

Coordinate expression of heme and globin is essential for effective erythropoiesis.

Erythropoiesis requires rapid and extensive hemoglobin production. Heme activates globin transcription and translation; therefore, heme synthesis must precede globin synthesis. As free heme is a potent inducer of oxidative damage, its levels within cellular compartments require stringent regulation. Mice lacking the heme exporter FLVCR1 have a severe macrocytic anemia; however, the mechanisms that underlie erythropoiesis dysfunction in these animals are unclear. Here, we determined that erythropoiesis failure occurs in these animals at the CFU-E/proerythroblast stage, a point at which the transferrin receptor (CD71) is upregulated, iron is imported, and heme is synthesized--before ample globin is produced. From the CFU-E/proerythroblast (CD71(+) Ter119(-) cells) stage onward, erythroid progenitors exhibited excess heme content, increased cytoplasmic ROS, and increased apoptosis. Reducing heme synthesis in FLVCR1-defient animals via genetic and biochemical approaches improved the anemia, implying that heme excess causes, and is not just associated with, the erythroid marrow failure. Expression of the cell surface FLVCR1 isoform, but not the mitochondrial FLVCR1 isoform, restored normal rbc production, demonstrating that cellular heme export is essential. Together, these studies provide insight into how heme is regulated to allow effective erythropoiesis, show that erythropoiesis fails when heme is excessive, and emphasize the importance of evaluating Ter119(-) erythroid cells when studying erythroid marrow failure in murine models.

Germline ETV6 mutations in familial thrombocytopenia and hematologic malignancy.

We report germline missense mutations in ETV6 segregating with the dominant transmission of thrombocytopenia and hematologic malignancy in three unrelated kindreds, defining a new hereditary syndrome featuring thrombocytopenia with susceptibility to diverse hematologic neoplasms. Two variants, p.Arg369Gln and p.Arg399Cys, reside in the highly conserved ETS DNA-binding domain. The third variant, p.Pro214Leu, lies within the internal linker domain, which regulates DNA binding. These three amino acid sites correspond to hotspots for recurrent somatic mutation in malignancies. Functional studies show that the mutations abrogate DNA binding, alter subcellular localization, decrease transcriptional repression in a dominant-negative fashion and impair hematopoiesis. These familial genetic studies identify a central role for ETV6 in hematopoiesis and malignant transformation. The identification of germline predisposition to cytopenias and cancer informs the diagnosis and medical management of at-risk individuals.

Mice lacking the sodium-dependent phosphate import protein, PiT1 (SLC20A1), have a severe defect in terminal erythroid differentiation and early B cell development.

Phosphate is critical in multiple biological processes (phosphorylation reactions, ATP production, and DNA structure and synthesis). It remains unclear how individual cells initially sense changes in phosphate availability and the cellular consequences of these changes. PiT1 (or SLC20A1) is a constitutively expressed, high-affinity sodium-dependent phosphate import protein. In vitro data suggest that PiT1 serves a direct role in mediating cellular proliferation; its role in vivo is unclear. We have discovered that mice lacking PiT1 develop a profound underproduction anemia characterized by mild macrocytosis, dyserythropoiesis, increased apoptosis, and a near complete block in terminal erythroid differentiation. In addition, the animals are severely B cell lymphopenic because of a defect in pro-B cell development and mildly neutropenic. The phenotype is intrinsic to the hematopoietic system, is associated with a defect in cell cycle progression, and occurs in the absence of changes in serum phosphate or calcium concentrations and independently of a change in cellular phosphate uptake. The severity of the anemia and block in terminal erythroid differentiation and B cell lymphopenia are striking and suggest that PiT1 serves a fundamental and nonredundant role in murine terminal erythroid differentiation and B cell development. Intriguingly, as the anemia mimics the ineffective erythropoiesis in some low-grade human myelodysplastic syndromes, this murine model might also provide pathologic insight into these disorders.

Enzymes to die for: exploiting nucleotide metabolizing enzymes for cancer gene therapy.

Suicide gene therapy is an attractive strategy to selectively destroy cancer cells while minimizing unnecessary toxicity to normal cells. Since this idea was first introduced more than two decades ago, numerous studies have been conducted and significant developments have been made to further its application for mainstream cancer therapy. Major limitations of the suicide gene therapy strategy that have hindered its clinical application include inefficient directed delivery to cancer cells and the poor prodrug activation capacity of suicide enzymes. This review is focused on efforts that have been and are currently being pursued to improve the activity of individual suicide enzymes towards their respective prodrugs with particular attention to the application of nucleotide metabolizing enzymes in suicide cancer gene therapy. A number of protein engineering strategies have been employed and our discussion here will center on the use of mutagenesis approaches to create and evaluate nucleotide metabolizing enzymes with enhanced prodrug activation capacity and increased thermostability. Several of these studies have yielded clinically important enzyme variants that are relevant for cancer gene therapy applications because their utilization can serve to maximize cancer cell killing while minimizing the prodrug dose, thereby limiting undesirable side effects.